Dengue serotype 1 vaccine development: The live attenuated DEN1 vaccine candidate virus rDEN1del30 has been evaluated in Phase I clinical trials and was found to be safe and immunogenic at a dose of 1000 PFU. It is a promising vaccine candidate for inclusion in a tetravalent dengue vaccine formulation. Additional studies are in progress to address the number and timing of two doses of this vaccine candidate. The studies involve two doses of vaccine given at an interval of 4 or 6 months. The results will be reported pending completion. Trials to determine the human infectious dose 50 are planned.[unreadable] Dengue serotype 2 vaccine development: rDEN2/4del30(ME) is an attenuated chimeric dengue virus in which the prM and E structural proteins of the DEN4 candidate vaccine, rDEN4del30, have been replaced by those of the prototypic DEN2 NGC virus. The live attenuated DEN2 vaccine candidate virus rDEN2/4del30(ME) has been evaluated in Phase I clinical trials and found to be safe and immunogenic at a dose of 1000 PFU. It is a promising vaccine candidate for inclusion in a tetravalent dengue vaccine formulation. Additional studies are in progress to address the number and timing of two doses of this vaccine candidate. Studies involve two doses of vaccine given at an interval of 4 or 6 months. Results will be reported pending completion. Trials to determine the human infectious dose 50 are planned.[unreadable] Dengue serotype 3 vaccine development; rDEN3/4del30(ME) is an attenuated chimeric dengue virus in which the prM and E structural proteins of the DEN4 candidate vaccine rDEN430 have been replaced by those of a DEN3 wild type virus. rDEN3/4del30(ME) or placebo was evaluated at a dose of 1,000 or 100,000 PFU in 28 healthy dengue-naive adult volunteers per dose. Studies are in progress, but less than 40% of vaccinees were infected with either dose indicating this vaccine candidate is not sufficiently infectious to be used as a component of a tetravalent dengue vaccine formulation. Two additional DEN3 vaccine candidates have been generated for trials in humans in FY09. [unreadable] Dengue serotype 4 vaccine development: rDEN4del30-200,201 is a live attenuated DENV-4 vaccine candidate specifically designed to further attenuate the rDEN4del30 parent virus. In a prior study, 5 of 20 vaccinees who received rDEN4del30 at 100,000 PFU developed moderately elevated levels of serum alanine aminotransferase (ALT). Mutational analysis of DENV-4 revealed that the 200,201 mutation in the NS5 polymerase specified greatly restricted virus replication in human hepatocarcinoma HuH-7 cells. In pre-clinical animal studies, the rDEN4del30-200,201 vaccine candidate was shown to be more attenuated than rDEN4del30 in both SCID-HuH-7 mice and rhesus monkeys. We compared the frequency and severity of solicited adverse events and level of viremia between recipients of 100,000 PFU of rDEN4del30-200,201 and recipients of 100,000 PFU of rDEN4del30, administered in a previous vaccine trial. Most notably, no volunteers vaccinated with rDEN4del30-200,201 had detectable viremia compared with 70% of recipients of rDEN4&#8710;30, a finding similar to that observed in rhesus monkeys. rDEN4del30-200,201 induced a dengue-like rash in fewer volunteers than the parent vaccine rDEN4del30. In addition, no volunteer who received rDEN4del30-200,201 developed an ALT elevation above the upper limit of the laboratory normal compared with 25% volunteers who received rDEN4del30. rDEN4&#8710;30-200,201 infected all vaccinees as evidenced by a 100% seroconversion rate. The decreased clinical signs and decreased replication confirmed that rDEN4del30-200,201 was more attenuated than its rDEN4del30 parent virus. The level of neutralizing antibody induced by rDEN4del30-200,201 at study day 42 was only slightly lower than that induced by rDEN4del30, a surprisingly insignificant trade-off considering the decreased reactogenicity. The abrogation of mild hepatotoxicity by the 200,201 mutation suggests that this mutation restricts replication of the vaccine in the liver of vaccine recipients. The restricted replication of rDEN4del30-200,201 in SCID HuH-7 mice and rhesus monkeys was also observed in humans, validating the pre-clinical approach of identifying a mutant virus with a decreased level of replication in human liver cells transplanted into SCID mice as a virus that is attenuated for humans. rDEN4del30-200,201 is a promising candidate for inclusion in a tetravalent DENV vaccine. We currently consider the rDEN4del30 vaccine our leading candidate for inclusion in the tetravalent vaccine for two reasons. At a proposed dose of 1000 PFU, it is economical to produce and has an acceptable safety profile; and, rDEN4del30 appears slightly more immunogenic in humans than rDEN4del30-200,201; this greater immunogenicity likely would translate into a more durable immunity. However, the present results with rDENdeldel30-200,201 are very encouraging because they identify a valuable backup vaccine candidate if rDEN4del30 has unanticipated adverse reaction upon further testing in humans and the results demonstrate that it is feasible to use genetic techniques to introduce defined mutations into the dengue virus genome to abrogate a specific adverse effect, in this instance, hepatotoxicity. A second candidate, rDEN4del30-4995, with decreased hepatotoxicity has also been made and evaluated in humans. These studies are in progress.[unreadable] Other dengue virus vaccine trials: Trials have been initiated to determine if infection with one dengue virus vaccine serotype modifies the replication and clinical response to a second serotype given after six months. This trial, in which four combinations of heterologous first and second vaccines are studied, has been initiated and largely enrolled.[unreadable] West Nile virus vaccine development trials in humans: Phase I trials are ongoing with a live attenuated West Nile virus vaccine candidate, WNVDEN4del30. Studies at 1000 and 100,000 infectious units per vaccinee indicated the vaccine was safe with low viremia, but was immunogenic in only 80% of vaccinees. To achieve a higher take rate, two studies are ongoing in which two doses of 10,000 infectious units of WNVDEN4del30 are given six months apart to one group and 100,000 infectious units are given six months apart to second group.[unreadable] Tick-borne encephalitis virus vaccine development trials in humans: With the steady rise in tick-borne encephalitis virus (TBEV) infections in Europe, development of a live attenuated vaccine that will generate long-lasting immunity would be of considerable benefit. A chimeric flavivirus, designated LGT/DEN4, was previously constructed to have a genome containing the prM and E protein genes of Langat virus (LGT), a naturally attenuated member of the TBEV complex, and the remaining genetic sequences derived from DEN4. LGT/DEN4 was highly attenuated in rodents and non-human primates and clinical trials in humans were initiated. Twenty-eight healthy seronegative adult volunteers were randomly assigned in a 4:1 ratio to receive 1000 PFU of LGT/DEN4 or placebo. LGT/DEN4 vaccine was safe and viremia was seen in only one vaccinee. Infection induced a neutralizing antibody response to wild-type LGT in 80% of volunteers with a geometric mean titer (GMT) of 1:63 present on day 42 post-immunization; but the antibody response against TBEV was much less frequent (35%) and lower in magnitude (GMT = 1:9). To assess the response to a booster dose, 21 of the original 28 volunteers were re-randomized to receive a second dose of either 1000 PFU of vaccine or placebo given 6 to 18 months after the first dose. The immunogenicity against either LGT or TBEV was not significantly enhanced after the second dose of vaccine. Thus, chimerization of LGT with DEN4 yielded a vaccine virus that was highly attenuated yet infectious in humans. The level of replication was sufficiently r